title: Substrate binding promotes formation of the Skp1-Cul1-Fbxl3 (SCF(Fbxl3)) protein complex.
abstract: The Skp1-Cul1-F-box protein (SCF) complex is one of the most well characterized types of ubiquitin ligase (E3), with the E3 activity of the complex being regulated in part at the level of complex formation. Fbxl3 is an F-box protein that is responsible for the ubiquitylation and consequent degradation of cryptochromes (Crys) and thus regulates oscillation of the circadian clock. Here we show that formation of the SCF(Fbxl3) complex is regulated by substrate binding in vivo. Fbxl3 did not associate with Skp1 and Cul1 to a substantial extent in transfected mammalian cells. Unexpectedly, however, formation of the SCF(Fbxl3) complex was markedly promoted by forced expression of its substrate Cry1 in these cells. A mutant form of Fbxl3 that does not bind to Cry1 was unable to form an SCF complex, suggesting that interaction of Cry1 with Fbxl3 is essential for formation of SCF(Fbxl3). In contrast, recombinant Fbxl3 associated with recombinant Skp1 and Cul1 in vitro even in the absence of recombinant Cry1. Domain-swap analysis revealed that the COOH-terminal leucine-rich repeat domain of Fbxl3 attenuates the interaction of Skp1, suggesting that a yet unknown protein associated with the COOH-terminal domain of Fbxl3 and inhibited SCF complex formation. Our results thus provide important insight into the regulation of both SCF ubiquitin ligase activity and circadian rhythmicity.
authors.affiliation: From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan and CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Kanae
authors.initials: K
authors.lastname: Yumimoto
authors.affiliation: From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan and CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Muneoka
authors.affiliation: From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan and CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Tsuboi
authors.affiliation: From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan and CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan. Electronic address: nakayak1@bioreg.kyushu-u.ac.jp.
authors.forename: Keiichi I
authors.initials: KI
authors.lastname: Nakayama
mesh_terms: D000818:Animals; D002940:Circadian Rhythm / Q000502:physiology*; D056931:Cryptochromes / Q000235:genetics / Q000378:metabolism; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology; D054875:Ubiquitination / Q000502:physiology
chemical_list: C538963:CRY1 protein, human; C538964:Cry1 protein, mouse; D056931:Cryptochromes; C100318:Cullin 1; D044842:Cullin Proteins; D044783:F-Box Proteins; C519795:FBXL3 protein, human; C519794:Fbxl3 protein, mouse; D046912:Multiprotein Complexes; D044786:S-Phase Kinase-Associated Proteins; C097351:SKP1 protein, human
keywords: Circadian Clock; Protein Assembly; Protein Degradation; Ubiquitin Ligase; Ubiquitylation